Genomic Analysis of Clostridium botulinum Group II by Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) was optimized for genomic analyses of Clostridium botulinum (nonproteolytic) group II. DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. A rapid (4-h) in situ DNA isolation method was also assessed and gave indistinguishable results. Genomic DNA from 21 strains of various geographical and temporal origins was digested with 15 rare-cutting restriction enzymes. Of these, ApaI, MluI, NruI, SmaI, and XhoI gave the most revealing PFGE patterns, enabling strain differentiation. Twenty strains yielded PFGE patterns containing 13 pulsotypes. From summation of MluI, SmaI, and XhoI restriction fragments, the genome size of C. botulinum group II was estimated to be 3.6 to 4.1 Mb (mean ± standard deviation = 3,890 ± 170 kb). The results substantiate that after problems due to DNases are overcome, PFGE analysis will be a reproducible and highly discriminating epidemiological method for studying C. botulinum group II at the molecular level.     

DNA Fingerprinting of Dairy Propionibacteria Strainsby Pulsed-Field Gel Electrophoresis

Restriction endonuclease patterns generated by Pulsed-Field Gel Electrophoresis (PFGE) were used to compare 96 strains of dairy propionibacteria originating from dairy products, international and industrial collections; endonucleases XbaI and SspI gave satisfactory restriction patterns. However, whereas XbaI can be used for Propionibacterium freudenreichii, SspI seems more suitable for the three other species: P. acidipropionici, P. thoenii, and P. jensenii. It is a convenient method to differentiate the dairy propionibacteria from closely related bacteria and from others usually present in dairy products. We observed a considerable restriction fragment length polymorphism among the Propionibacterium chromosomes and especially for P. freudenreichii: among 48 strains we detected 40 different patterns. This species is the most commonly encountered in the Swiss-type cheeses and is the only Propionibacterium species used as a cheese starter. Conversely, the species P. acidipropionici is not very diverse: among nine strains we observed only four different patterns, two of which were closely related. This is probably because this species is not used as a starter in cheese manufacture and consequently is poorly represented in collections. When strains come from geographical different isolates, their patterns are always different with very few common bands. The presence of numerous identical strains was due to the fact that they were present at the same time in the national collections, research laboratory collections, and in the industrial ones.     

Genomic DNA Fingerprinting of Oenococcus oeni Strains by Pulsed-Field Gel Electrophoresis and Randomly Amplified Polymorphic DNA-PCR

Genetic diversity of 60 Oenococcus oeni strains from different wines was evaluated by numerical analysis of (i) pulsed-field gel electrophoresis (PFGE) patterns with endonuclease ApaI and (ii) randomly amplified polymorphic DNA (RAPD)-PCR fingerprints with four oligonucleotide primers. Sixty-two percent of the strains could be distinguished by PFGE, whereas most strains were identified by distinct RAPD-PCR profiles and associated according to the geographical origin. Because of its rapidity and reliability, RAPD-PCR appeared to be a suitable method for typing and monitoring O. oeni strains in winemaking. Received: 3 November 1999 / Accepted: 8 December 1999     

Genomic Diversity within the Genus Pediococcus as Revealed by Randomly Amplified Polymorphic DNA PCR and Pulsed-Field Gel Electrophoresis

The genomic diversity of 33 previously assigned strains from six species within the genus Pediococcus was assessed by randomly amplified polymorphic DNA (RAPD) PCR and pulsed-field-gel electrophoresis (PFGE). The RAPD PCR patterns produced by two separate random primers, termed P1 (ACGCGCCCT) and P2 (ATGTAACGCC), were compared by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm. Pattern variations between repeat samples set a strain discrimination threshold of less than 70% similarity. P1 and P2 primers alone and in combination produced 14, 21, and 28 distinct patterns, respectively. When each strain was assigned with a type strain with which it shared the highest level of similarity, both primers grouped 17 of the 27 strains to their proposed species. PFGE following genomic digestion with the restriction enzymes ApaI, NotI, and AscI produced 30, 32, and 28 distinct macrorestriction patterns, respectively. Specific DNA fragments within the NotI and AscI macrorestriction patterns for each strain were observed that allowed 27 of the 33 strains to be assigned to their proposed species. For example, following digestion with AscI, all Pediococcus parvulus strains were characterized by two DNA fragments, one of approximately 220 kb and another between 700 and 800 kb. The exceptions correlated with those observed with both RAPD PCR primers and included three P. damnosus and two P. pentosaceus strains that grew at temperatures regarded as nonpermissive for their proposed species but not for those with which they grouped.     

Comparison of Leuconostoc oenos Strains by Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis of chromosomal DNA digested with NotI or SfiI was used to differentiate individual strains of Leuconostoc oenos. L. oenos isolates with 13 different restriction digest patterns were detected in New Zealand wines undergoing malolactic fermentation. The average genome size was estimated to be 1,800 kb.     

Identification and Characterization of Pathogenic Yersinia enterocolitica Isolates by PCR and Pulsed-Field Gel Electrophoresis

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n = 48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.     

Optimization of Pulsed-Field Gel Electrophoresis for Legionella pneumophila Subtyping

A total of 32 strains of Legionella pneumophila were used to optimize pulsed-field gel electrophoresis (PFGE) for subtyping of L. pneumophila. Twenty-six isolates of L. pneumophila with various origins and 11 isolates from five different water systems were used as the panels. For optimization of electrophoretic parameters (EPs) of SfiI PFGE, 26 isolates were analyzed with SfiI digestion, using four EPs yielding the same D value. The EP of a switch time of 5 to 50 s for 21 h had the smallest similarity coefficients and was declared the optimal EP for SfiI PFGE of L. pneumophila. By software analysis and pilot study, AscI was chosen as another PFGE enzyme. AscI PFGE could cluster the isolates from each water system into the same or very similar patterns and had a high degree of typing concordance with other molecular methods. In evaluating the discriminatory power of AscI with the panel of 26 isolates, AscI PFGE gave one single pattern and a D value of 100%. AscI PFGE had a high discriminatory power and a high degree of consistency with epidemiological data and other molecular typing methods for L. pneumophila subtyping, and hence, AscI could be used as a restriction enzyme in PFGE subtyping of L. pneumophila.Legionella pneumophila is an environmental organism that can cause disease in humans and is increasingly recognized as an important pathogen causing nosocomial pneumonia. Potable water systems (14, 26), spa water (28), and cooling towers (7, 13) are among the sources implicated in outbreaks of Legionnaires’ disease. Transmission of bacteria from the environment to humans occurs via inhalation or aspiration of Legionella-containing aerosols (3, 5). Strain differentiation is necessary for the identification of sources of contamination and determination of routes of transmission; this could in turn enable us to more accurately detect outbreaks and limit the spread of L. pneumophila infections. A variety of subtyping techniques have been used to identify and characterize L. pneumophila strains, including monoclonal antibody (MAb) analysis (16, 19), ribotyping (4), amplified fragment length polymorphism (AFLP) analysis (9, 22), PCR-based methods (15, 24), sequence-based typing (SBT) (9, 16), and pulsed-field gel electrophoresis (PFGE) (1, 6).Preliminary reports demonstrated that PFGE is a highly discriminative epidemiological marker for subtyping of L. pneumophila (6, 11, 23, 25), and a number of L. pneumophila PFGE protocols have been described in the literature (1, 2, 4, 14); however, most laboratories that use PFGE to subtype L. pneumophila cannot compare their results because the protocols differ from each other in critical parameters, such as the restriction enzymes and electrophoresis conditions used to generate the DNA fingerprints. To enhance our ability to monitor this pathogen, there is an urgent need for a standardized L. pneumophila PFGE protocol which can readily be implemented in different laboratories for information interpretation.An optimal PFGE protocol produces a suitable number of restriction fragments and gives distinct patterns by agarose gel electrophoresis, with these determined by the restriction enzymes and the electrophoretic parameters (EPs) used. SfiI is the most frequently used enzyme in conventional PFGE protocols for L. pneumophila, and there are several different EPs for SfiI digestion used by investigators for characterization and epidemiological studies. For a certain restriction enzyme, selection of the EP with the smallest similarity coefficients will increase the discriminatory power of PFGE. As the first phase of this study, we compared the similarity coefficients obtained for four EPs with SfiI digestion and determined the one with the maximal discriminatory power.There were some problems found in practical applications of epidemiological investigation of L. pneumophila by PFGE with single SfiI digestion, such as having epidemiologically unrelated strains exhibit the same patterns (30) and the appearance of “ghost” or “phantom” bands. Combination use of two enzymes would give a higher discriminatory power and more accurate results (10, 29). Thus, as the second phase of this study, we selected another suitable enzyme and compared it with SfiI to evaluate the possibility of its use in characterization and epidemiological studies of L. pneumophila.     

Genotyping of Staphylococcus epidermidis by Small-Fragment Restriction Endonuclease Analysis and Pulsed-Field Gel Electrophoresis of Genomic Restriction Fragments

Small-fragment restriction endonuclease analysis (SF-REA) was established as a typing tool for Staphylococcus epidermidis. A total of 60 isolates comprising 48 epidemiologically nonrelated strains and 12 putatively linked isolates from 7 patients in 2 wards were analyzed. Nonrelated isolates were characterized by unique fingerprints when DNA was cleaved with EcoRI or ClaI, electrophoretically separated in a polyacrylamide gel, and silver stained. Three blood culture isolates from one patient in an intensive care unit, 4 isolates obtained from a child over a span of 2 weeks, and 5 isolates from 5 newborns in the same ward were grouped into 3 DNA pattern types, indicating identity of sequential isolates from 2 patients and nosocomial transmission of one Staphylococcus epidermidis strain between 5 babies. Results from pulsed-field gel electrophoresis of SmaI and SacII DNA digests and conventional marker systems such as antibiogram and plasmid profile were in accordance with these interpretations, whereas slight variation was observed in the biotypes of several strains. From the results of this study, we conclude that SF-REA is a precise and efficient method for the genotypic characterization of Staphylococcus epidermidis strains that can be used as a rapid and reliable typing tool.     

Diversity of Proteolytic Clostridium botulinum Strains, Determined by a Pulsed-Field Gel Electrophoresis Approach

Pulsed-field gel electrophoresis (PFGE) was applied to the study of the similarity of 55 strains of proteolytic Clostridium botulinum (C. botulinum group I) types A, AB, B, and F. Rare-cutting restriction enzymes ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI were tested for their suitability for the cleavage of DNA of five proteolytic C. botulinum strains. Of these enzymes, SacII, followed by SmaI and XhoI, produced the most convenient number of fragments for genetic typing and were selected for analysis of the 55 strains. The proteolytic C. botulinum species was found to be heterogeneous. In the majority of cases, PFGE enabled discrimination between individual strains of proteolytic C. botulinum types A and B. The different toxin types were discriminated at an 86% similarity level with both SacII and SmaI and at an 83% similarity level with XhoI. Despite the high heterogeneity, three clusters at a 95% similarity level consisting of more than three strains of different origin were noted. The strains of types A and B showed higher diversity than the type F organisms which formed a single cluster. According to this survey, PFGE is to be considered a useful tool for molecular epidemiological analysis of proteolytic C. botulinum types A and B. However, epidemiological conclusions based on PFGE data only should be made with discretion, since highly similar PFGE patterns were noticed, especially within the type B strains.     

Comparison of Pulsed-Field Gel Electrophoresis DNA Fingerprints of Field Isolates of the Entomopathogen Bacillus popilliae

Eight strains of the entomopathogen Bacillus popilliae were analyzed by pulsed-field gel electrophoresis, and genomic size estimates of ^sim2,600 to 3,500 kb were obtained. The type strain, ATCC 14706, had a genomic size of 3,395 kb. For the six New Zealand isolates, the degree of similarity in the pulsed-field gel electrophoresis fingerprints may correlate with the geographical closeness of the sites of isolation. The plasmid profiles of the New Zealand isolates were also compared; four of the six strains carry plasmids in the 3.6- to 9.7-kb size range.     

Meta-Analysis of Pulsed-Field Gel Electrophoresis Fingerprints Based on a Constructed Salmonella Database

A database was constructed consisting of 45,923 Salmonella pulsed-field gel electrophoresis (PFGE) patterns. The patterns, randomly selected from all submissions to CDC PulseNet during 2005 to 2010, included the 20 most frequent serotypes and 12 less frequent serotypes. Meta-analysis was applied to all of the PFGE patterns in the database. In the range of 20 to 1100 kb, serotype Enteritidis averaged the fewest bands at 12 bands and Paratyphi A the most with 19, with most serotypes in the 13−15 range among the 32 serptypes. The 10 most frequent bands for each of the 32 serotypes were sorted and distinguished, and the results were in concordance with those from distance matrix and two-way hierarchical cluster analyses of the patterns in the database. The hierarchical cluster analysis divided the 32 serotypes into three major groups according to dissimilarity measures, and revealed for the first time the similarities among the PFGE patterns of serotype Saintpaul to serotypes Typhimurium, Typhimurium var. 5-, and I 4,[5],12:i:-; of serotype Hadar to serotype Infantis; and of serotype Muenchen to serotype Newport. The results of the meta-analysis indicated that the pattern similarities/dissimilarities determined the serotype discrimination of PFGE method, and that the possible PFGE markers may have utility for serotype identification. The presence of distinct, serotype specific patterns may provide useful information to aid in the distribution of serotypes in the population and potentially reduce the need for laborious analyses, such as traditional serotyping.     

Determination of Gardnerella vaginalis Genome Size by Pulsed-Field Gel Electrophoresis

The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size.     

Genome Analysis of Several Marine, Magnetotactic Bacterial Strains by Pulsed-Field Gel Electrophoresis

The genomic DNA of three strains of marine magnetotactic bacteria, including two facultatively anaerobic vibrios, strains MV-1 and MV-2, and the microaerophilic coccus, strain MC-1, was analyzed by pulsed-field gel electrophoresis (PFGE). Digestion of the genomic DNA of strain MV-1 by the restriction endonucleases AvrII, BamHI, HindIII, NheI, SalI, SfiI, SgfI, SgrAI, and XbaI resulted in a large number of fragments below 400 kb that were difficult to resolve by PFGE. Digestion of MV-1 DNA with NotI and RsrII resulted in no fragments. Treatment of genomic DNA of strains MV-1 and MV-2 with PacI, PmeI, and SpeI yielded a manageable number of fragments (ca. 20) that were relatively easily resolved with PFGE, while PacI and SpeI were effective for strain MC-1. There was no evidence for the presence of plasmids and linear chromosomes in any of the strains, and strains MV-1 and MV-2 appear to contain a single, circular chromosome. Genome sizes of strains MV-1, MV-2, and MC-1 were estimated to be between 3.6 and 3.9 Mb (mean ± SD; 3.7 ± 0.2), 3.3 and 3.7 Mb (3.6 ± 0.2), and 4.3 and 4.7 Mb (4.5 ± 0.3), respectively. The restriction fragment patterns of the vibrioid strains MV-1 and MV-2 were extremely similar, suggesting that the strains are closely related. Received: 30 March 1999 / Accepted: 17 May 1999     

Genetic Fingerprinting of Brevibacterium linens by Pulsed-Field Gel Electrophoresis and Ribotyping

Members of Brevibacterium linens display physiological features that are relevant for cheese production. The genomes of five B. linens strains deposited on culture collections were compared by examining large restriction fragments on pulsed-field gel electrophoresis and detection of polymorphism at the level of 16S rRNA genes. Pulsed-field analysis with the endonucleases DraI and AsnI showed a characteristic restriction profile for each strain and allowed the calculation of genome sizes ranging between 3.2 and 3.9 Mbp. No linear genomic elements were detected. Polymorphisms at the level of 16S rRNA genes were revealed by hybridization with an oligonucleotide probe complementary to a universal domain of the 16S genes. An EcoRI fragment of 1.4 kb was identified as common to all strains under study. According to the number of positive bands detected by the probe, at least four rRNA operons must be present on the genome of the B. linens strains here studied. Received: 13 January 2000 / Accepted: 9 February 2000     

Pulsed-Field Gel Electrophoresis Is More Discriminating Than Multilocus Enzyme Electrophoresis and Random Amplified Polymorphic DNA Analysis for Typing Pyogenic Streptococci

The SmaI restriction endonuclease digestion patterns of chromosomal DNAs from 99 pyogenic streptococci belonging to Lancefield group A (41 Streptococcus pyogenes), group C (seven S. dysgalactiae, 11 \QS. equisimilis\W, three S. equi, eight S. zooepidemicus) and group G (25 human group G Streptococcus, four S. canis) were analyzed by pulsed-field gel electrophoresis (PFGE), and the results were compared with those previously obtained by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA analysis (RAPD). PFGE revealed 93 distinct types among the 99 strains, and no patterns were common to strains of different species. The discriminatory power of PFGE was greater than that of MLEE and RAPD for groups A and G streptococci. The polymorphism among group C streptococci was similar with the three techniques. PFGE is, therefore, the most efficacious method for epidemiological typing of pyogenic streptococci. Received: 31 July 1996 / Accepted: 17 August 1996     

Genome Sizes of Desulfovibrio desulfuricans, Desulfovibrio vulgaris, and Desulfobulbus propionicus Estimated by Pulsed-Field Gel Electrophoresis of Linearized Chromosomal DNA

Pulsed-field gel electrophoresis (PFGE) of linearized, full-length chromosomal DNA was used to estimate the genome sizes of three species of sulfate-reducing bacteria. Genome sizes of Desulfovibrio desulfuricans, Desulfovibrio vulgaris, and Desulfobulbus propionicus were estimated to be 3.1, 3.6, and 3.7 Mb, respectively. These values are double the genome sizes previously determined for two Desulfovibrio species by two-dimensional agarose gel electrophoresis of DNA cut with restriction enzymes. PFGE of full-length chromosomal DNA could provide a generally applicable method to rapidly determine bacterial genome size and organization. Received: 1 October 1996 / Accepted: 5 November 1996